Methods of the invention can be used to analyze complex mixtures of nucleic acids in small amounts of source material. Common applications are the quantification of messenger RNA levels for specific genes in an organism or tissue or the determination of the allele status of genetic polymorphisms in genomic DNA. Often there is a requirement to analyze many different nucleic acid sequences in a single sample, and it is often necessary to amplify the target nucleic acids or signal molecules to obtain detectable signals in an assay.
DNA microarrays provide the ability to analyze many target sequences in a sample. However, conventional microarray analysis is limited by the cost and difficulty of preparing large numbers of target molecules from limited amounts of sample and by poor hybridization specificity. Multiplexed analysis—amplifying target sequences or target-specific probe molecules from many targets in a single sample—can minimize the cost of reagents and the consumption of precious samples. Methods with better sequence specificity than simple hybridization reactions—e.g., analytical methods based on activities of nucleic acid modifying enzymes—provide improved reliability and accuracy of the quantification of specific sequences and the detection of specific polymorphisms.
Multiplex polymerase chain reactions (PCR) have been used to decrease the sample preparation for scoring genetic variation by hybridization on DNA microarrays. Unfortunately many of the target sequences fail to amplify optimally, especially when large multiplex factors (large numbers of target sequences amplified simultaneously) are used (Wang et al., Science 280:1077, 1998). Multiplex PCR has been performed with 5′ extensions or “tails” on target-specific PCR primers which incorporate universal priming sequences into amplification products (Brownie et al, Nucleic Acids Research 25:3235, 1997; U.S. Pat. No. 5,858,989; Jeffreys et al; Favis et al, Nature Biotechnology 18:561, 2000). Amplification is performed for a limited number of cycles with these tailed target-specific primers, and then further amplification is performed with high concentrations of universal primers with the same sequence as the 5′ tails of the target-specific primers.
Multiplex amplification with universal primers after appending 5′ extensions on amplification products with adapter primers has been used with strand displacement amplification (U.S. Pat. No. 5,422,252, Walker et al.). Generic or universal primers have been used to amplify ligatable probes (U.S. Pat. No. 5,876,924, Zhang et al.; Thomas et al., Arch Pathol Lab Med 123:1170, 1999). The use of generic primers for probe amplification is advantageous compared to convention target amplification with PCR, since primer binding and amplification is not subject to the variability of target sequences. The ligatable probes may be pairs of linear probes or a single circularizable probe. In the case of pairs of linear probes the generic primer sequences are incorporated into the 3′ and 5′ ends of the probes, respectively (U.S. Pat. No. 5,876,924, Zhang et al.). In the case of circularizable probes, generic primer sequences are incorporated into the linker region between the target-specific termini of the probe. Amplification of circularizable probes may be performed with a single generic primer (“rolling circle amplification”, RCA) or with a pair of generic PCR primers (U.S. Pat. No. 5,876,924, Zhang et al.; Thomas et al., Arch Pathol Lab Med 123:1170, 1999). Amplified circularizable probes are attractive alternatives to PCR for multiplexed analysis (Isaksson and Landegren, Curr Opin Biotechnol. 10:11, 1999). Ligation reaction provide excellent sequence discrimination for scoring SNPs (Thomas et al., Arch Pathol Lab Med 123:1170, 1999; Favis et al, Nature Biotechnology 18:561, 2000).
Another type of reaction that provides excellent sequence discrimination and that also provides amplification of the probes is the Invader assay developed by the Third Wave Technologies Company (U.S. Pat. No. 5,846,717; U.S. Pat. No. 5,888,780; U.S. Pat. No. 5,985,557; U.S. Pat. No. 5,994,069; U.S. Pat. No. 6,001,567). With this method pairs of probes are hybridized to nucleic acid targets, and an endonuclease enzyme effects cyclic structure-dependent cleavage of one of the probes, if the probe matches the target sequence. A fragment of arbitrary sequence with a ligatable 3′ terminus (“flap”) is cleaved from the probe by the enzyme. The reaction provides substantial amplification and high specificity for scoring of SNPs (Ryan et al., Mol Diagn 4:135, 1999, Griffin et al., Proc Natl Acad Sci USA 96:6301, 1999) and has great potential for multiplexing the analysis of many targets in a single sample (Griffin et al., Proc Natl Acad Sci USA 96:6301, 1999).
Capture of target sequences on microarrays of nucleic acid probes is limited by less than optimal discrimination between related but non-identical sequences and by the secondary structure of target molecules. Analysis of single nucleotide polymorphisms (SNPs) by hybridization on oligonucleotide arrays is error prone and requires the use of highly redundant sets of probes (Wang et al., Science 280:1077, 1998; Cargill et al., Nature Genetics 22: 231, 1999). A method of enhanced oligonucleotide capture uses partially duplex probes and enzymatic reactions (ligation and/or primer extension) to accurately discriminate perfectly matched targets from those containing mismatches (Broude et al., Proc. Natl. Acad. Sci. 91: 3072, 1994; U.S. Pat. No. 5,503,980, Cantor; Gunderson et al., Genome Research 8:1142, 1998). Analysis of the efficiency of hybridization of various nucleic acid sequences (Mir and Southern, Nature Biotechnology 17:788, 1999) has demonstrated that the hybridization is most efficient at single stranded regions near duplex stems, probably due to the presence of helical order and the absence of tertiary interactions. Ligation and polymerase reactions have been shown to provide excellent discrimination of perfectly and mismatched probes required for the scoring of SNPs (Thomas et al., Arch Pathol Lab Med 123:1170, 1999; Favis et al, Nature Biotechnology 18:561, 2000; Pastinen, et al., Genome Research 7:606, 1997). Thus capture of nucleic acid targets on partially duplex probes coupled with enzymatic discrimination provides for very high specificity. The probes capture only terminal sequences on target molecules; internal target sequences that match the single stranded overhang sequences of the probes are not captured.